Morphological variation and nutrition of a new monocentric marine fungus

Summary The life cycle of a new marine Phycomycete, Thraustochytrium aureum, is described based on its development on pollen in sea water. Comparisons are made with the amoeboid habit of the fungus on nutrient agar and an hypothesis as to the cause of the phenomenon is discussed. Inoculation studies suggest that T. aureum may be primarily a saprophyte of vascular plant debris. The fungus is obligately marine and apparently euryhaline. Maximal growth occurs with 2.0% NaCl not replaceable by KCl. The omission of KCl from media does not affect yields. The salinity optimum does not vary with temperature and the organism grows best at 12–25°C. It can utilize the nitrogen in (NH₄)₂SO₄ but optimal harvests are obtained with glutamate as the source of that element and with carbon supplied as glucose, maltose, or soluble starch.     

Effects of sodium chloride on growth of ectomycorrhizal fungal isolates in culture

We studied the tolerance of ectomycorrhizal (ECM) fungi to sodium chloride (NaCl) to find the best fungus to aid growth of Pinus thunbergii. Four ECM fungi, Cenococcum geophilum, Pisolithus tinctorius, Rhizopogon rubescens, and Suillus luteus, were grown in liquid MMN media with five different concentrations of NaCl for 30 days, and their mycelial weights were determined. Mycelial weights of P. tinctorius and R. rubescens were not significantly different between 0 mM and 200 mM, whereas those of C. geophilum and S. luteus decreased with increasing NaCl concentration, indicating that the former two species were more tolerant to higher NaCl concentrations than the latter species. We further studied the intraspecific differences in NaCl tolerance of nine P. tinctorius isolates. They were grown on MMN agar media with six different concentrations of NaCl for 21 days, and their radial growth was measured. In total, the hyphal growth at 25 mM NaCl was significantly higher than those at the other NaCl concentrations, and EC₅₀ values were confirmed at between 50 mM and 200 mM. Among the isolates, Pt03 and Pt21 showed measurable growth at 200 mM; the growth of Pt03 was not significantly different between 0 mM and 200 mM. The results indicate that there are intraspecific variations in NaCl tolerance of Pisolithus species.     

Osmotic responses of unicellular blue-green algae (cyanobacteria): changes in cell volume and intracellular solute levels in response to hyperosmotic treatment

Abstract Changes in cell volume and solute content upon hyperosmotic shock have been studied for six unicellular blue-green algae (cyanobacteria): Synechococcus PCC 6301, PCC 6311; Synechocystis PCC 6702, PCC 6714, PCC 6803 and PCC 7008. The extent of change in volume was shown to be dependent upon the solute used to establish the osmotic gradient, with cells in NaCl showing a reduced shrinkage when compared to cells in media containing added sorbitol and sucrose. Uptake of extracellular solutes during hyperosmotic shock was observed in Synechocystis PCC 6714, with maximum accumulation of external solutes in NaCl and minimum solute uptake in sucrose solutions. Conversely, solute loss from the cells (K⁺ and amino acids) was greatest in sucrose-containing media and least in NaCl. The results show that these blue-green algae do not behave as ‘ideal osmometers’ in media of high osmotic strength. It is proposed that short-term changes in plasmalemma permeability in these organisms may be due to transient membrane instability resulting from osmotic imbalance between the cell and its surrounding fluid at the onset of hyperosmotic shock.     

Relationship between carbohydrate movement and the symbiosis in lichens with green algae

Summary When isolated in pure culture, four genera of lichen algae were able to produce the polyol which is known to move from the alga to the fungus in lichens with these algae. This conclusion corrects earlier suggestions that the mobile polyol is only formed by the alga in the lichen thallus. Stichococcus produced sorbitol and it is therefore suggested that, in lichens with this alga, sorbitol moves between the symbionts. Hyalococcus and Stichococcus had a similar pattern of incorporation of H¹⁴CO ₃ ^- in the light, suggesting a close relationship between these algae which are only separated now on morphological grounds.The pattern of incorporation of H¹⁴CO ₃ ^- in the light into Cladonia cristatella and its alga (Trebouxia erici) in culture indicates that in the cultured algae more ¹⁴C was incorporated into ethanol insoluble substances and lipids and less into ribitol than in the lichen. The pattern in a joint culture of the alga and the fungus of C. cristatella was approximately intermediate between that of the lichen and the alga. However, only a small amount of ¹⁴C fixed by the alga reached the fungus in the joint culture, and it is therefore suggested that the presence of the fungus without morphological differentiation into a lichen thallus is not sufficient to promote the alga to release carbohydrate.     

Response of Scenedesmus Incrassatulus to Salt Stress as Affected by Methyl Jasmonate

Exposure of the freshwater green alga Scenedesmus incrassatulus Bohl, strain R-83 to salt stress (175 mM NaCl) resulted in a reduction of its growth and ¹⁴CO₂ fixation and in an increase of accumulation of free proline and malondialdehyde (MDA). The accumulation of proline in the light was higher than in dark. NaCl significantly inhibited the Fe-induced release of organic chelators from the cells. Exogenously supplied 10^–4M methyl jasmonate (JA-Me) did not considerably change the ¹⁴CO₂ fixation, but increased proline and MDA accumulation in the cells and moderately inhibited the release of chelators from cells. JA-Me supplied simultaneously with NaCl helps the algae to counteract the salt stress.     

INFLUENCE OF SALINITY ON THE FORM OF ASTEROCYTIS IN PURE CULTURE1

The growth form of an isolate of Asterocytis ornata in pure culture changed from filamentous in a full-strength seawater medium to a unicellular or bicellular form closely resembling the genus Chroothece in a medium of quarter-strength seawater. The alga did not grow in media of lower salinities, or in media in which seawater was replaced by KCl, NaCl, or CaCl₂. The formation of filaments from single cells could not be attributed to osmolarity. The controlling factors seemed to be the concentrations of cations, notably Na⁺ and Mg⁺⁺. However, the filaments produced experimentally in a defined medium, were never as extensively developed as those occurring in seawater, an indication that further studies should be done. The occurrence of the Asterocytis form, in freshwater habitats remains to be explained. There may be distinct physiological races, if not true species, within the genus.     

Paxillus involutus mycorrhiza attenuate NaCl-stress responses in the salt-sensitive hybrid poplar Populus×canescens

In order to characterise the effect of ectomycorrhiza on Na⁺-responses of the salt-sensitive poplar hybrid Populus × canescens, growth and stress responses of Paxillus involutus (strain MAJ) were tested in liquid cultures in the presence of 20 to 500 mM NaCl, and the effects of mycorrhization on mineral nutrient accumulation and oxidative stress were characterised in mycorrhizal and non-mycorrhizal poplar seedlings exposed to 150 mM NaCl. Paxillus involutus was salt tolerant, showing biomass increases in media containing up to 500 mM NaCl after 4 weeks growth. Mycorrhizal mantle formation on poplar roots was not affected by 150 mM NaCl. Whole plant performance was positively affected by the fungus because total biomass was greater and leaves accumulated less Na⁺ than non-mycorrhizal plants. Energy dispersive X-ray microanalysis using transmission electron microscopy analysis of the influence of mycorrhization on the subcellular localisation of Na⁺ and Cl⁻ in roots showed that the hyphal mantle did not diminish salt accumulation in root cell walls, indicating that mycorrhization did not provide a physical barrier against excess salinity. In the absence of salt stress, mycorrhizal poplar roots contained higher Na⁺ and Cl⁻ concentrations than non-mycorrhizal poplar roots. Paxillus involutus hyphae produced H₂O₂ in the mantle but not in the Hartig net or in pure culture. Salt exposure resulted in H₂O₂ formation in cortical cells of both non-mycorrhizal and mycorrhizal poplar and stimulated peroxidase but not superoxide dismutase activities. This shows that mature ectomycorrhiza was unable to suppress salt-induced oxidative stress. Element analyses suggest that improved performance of mycorrhizal poplar under salt stress may result from diminished xylem loading of Na⁺ and increased supply with K⁺.     

NUTRITION OF THE GREEN ALGA GOLENKINIA

Defined media were established for four strains of Golenkinia. Strains 929 and 930 require thiamine and vitamin B₁₂, strain 931 requires the latter and is stimulated by thiamine; only strain 320 and a mutant of strain 931 are capable of completely autotrophic growth. The optimal nitrogen concentration is 0.025 m except for strain 931 where it is 0.015 m and the requirement may be met by nitrate, ammonium, or urea. Optimal levels of the other components are: 0.0025 m KH₂PO₄, 0.0005 to 0.001 m MgSO₄, and 50 ml of a stock trace-element solution per liter. Only strain 931 required calcium; however, its addition stimulates the growth of strains 929 and 930. The optimal pH before autoclaving is 6.8. Maximal growth rates of two to three cell doublings per day and yields of 26 to 30 (log₂) cells per ml were obtained. These growth data compare favorably with those for other unicellular green algae.     

Induced tolerance of Sclerotium cepivorum to antibiotics in the presence of onion exudates

The onion white rot pathogen Sclerotium cepivorum was cultured on agar media containing 2% malt extract and one of the antifungal antibiotics, endomycin, griseofulvin, venturicidin and cycloheximide at concentrations that reduced but did not prevent growth of mycelium. When onion seeds or agar discs impregnated with diffusates from onion bulbs were placed on the antibiotic media, radial growth of the fungus was greatly increased, and there was a profuse development of aerial mycelium. Gaseous diffusates from onion tissue and from impregnated agar discs were also effective. On the antibiotic media, tomato, cabbage and radish seeds did not stimulate the growth of S. cepivorum and the onion exudates did not stimulate the growth of four other fungi. This and other evidence is considered to show that the stimulation of growth of S. cepivorum was not caused by any direct effect on the antibiotics but by a tolerance of the fungus to them, which was specifically induced by an exudate from its host plant. The phenomenon may be related to the reported reversal by onion extracts of the inhibitory effects of soil mycostasis on germination of sclerotia of the fungus.     

A SEAWATER MEDIUM FOR DINOFLAGELLATES AND THE NUTRITION OF CACHONINA NIEI1

Two media have been devised: an enriched seawater medium for culture of dinoflagellates and a defined medium for rapid growth of the dinoflagellate Cachonina niei. A wide range in salinity (10.23–42.38 g/liter NaCl) is tolerated by C. niei. Below 0.6 g/liter MgSO₄, 0.19 g/liter KCl, and 0.22 g/liter CaCl₂, the generation time greatly increases. Increase in MgSO₄ to 7.22 g/liter, KCl to 1.12 g/liter or CaCl₂ to 2.22 g/liter has little effect on generation time. The temperature optimum is 19–23 C. Saturating light intensity for growth is 1000 ft-c and for photosynthesis (determined manometrically) is slightly less than 2000 ft-c. Cachonina niei requires B₁₂ and thiamin. Neither silicate nor its competitive inhibitor germanate affects generation time or cell yield indicating silicon is not required. Of a variety of buffers tested, Tris is the best. Optimal growth occurs at pHs of 7.5–8.3. Glycerol is inhibitory and does not support dark growth.     

Nutritional study on <Emphasis Type="Italic">Embellisia astragali</Emphasis>, a fungal pathogen of milk vetch (<Emphasis Type="Italic">Astragalus adsurgens</Emphasis>)

Embellisia astragali is a strong, virulent pathogen that develops within milk vetch (Astragalus adsurgens). In order to determine nutrient requirements, the fungus was cultured on 9 carbon sources, 9 nitrogen sources, and 13 growth media in the dark at 25°C. Growth rates and sporulation capacity were measured after 4 and 12 weeks. All carbon sources supported growth, but only soluble starch, inulin, and dextrose supported sporulation. In general, better growth was obtained on disaccharides and polysaccharides than on monosaccharides. Compared with no growth on NH₄ ⁺-N and urea, the fungus grew little on all NO₃ ⁻-N, amino-N, and other organic-N such as peptone. There was no sporulation or very sparse conidia on almost all nitrogen sources with supplied dextrose or soluble starch as sole carbon source. The better growth and sporulation on most of the semidefined media than on defined media indicates that some components in plant or animal material may be vital to the fungus. Sporulation was positively correlated with growth rate in N source experiment at 12 weeks and in growth media experiment at 4 and 12 weeks. The fungus favors grow within agar with growth rate less than 1.18 mm day⁻¹.     

Determination of the Chemical Form of Fluorine in Tea Infusions by 19F-NMR

A novel marine ice-nucleating bacterium, KUIN-5, was isolated from a marine algae, Monostroma latissum. Strain KUIN-5 was identified as a Pseudomonas sp. from its characteristics and taxonomies; the optimum temperature and pH for its growth were 25°C and 6.0, respectively. When strain KUIN-5 was aerobically cultured in Carlucci-Pramer medium (pH 6.0) for 50 h at 25°C, the highest ice-nucleating activity of the cells among the media for marine bacteria was obtained, and the ice-nucleating temperature, T₅₀, was indicated to be ? 3.2°C. Also, the optimum concentration of NaCl for the growth in this medium, which was prepared with distilled water instead of seawater, was 2.0% (w/v) and then the ice-nucleating activity was inversely proportional to the NaCl concentration. Moreover, when strain KUIN-5 was cultured in Davis medium under optimum conditions, it produced insoluble polysaccharide (IPS) in the culture. The maximum amount of IPS production by strain KUIN-5 was 84.5 mg/ml of medium under optimum conditions. Therefore, this IPS was isolated and could be identified as cellulose, based on TLC or HPLC of the acid hydrolysate, and GC-MS of the acetylated polyalcohol prepared by periodate oxidation and Smith degradation of this polysaccharide. This is the first report of cellulose production by a marine ice-nucleating bacterium.     

Studies on toxigenic fungi in roasted foodstuff (Salted seed) and halotolerant activity of emodin-producingAspergillus wentii

Commercial roasted salted peanuts (3% NaCl), popcorn (1% NaCl), summer-squash (9% NaCl), sunflower (3% NaCl) and wild-melon (3% NaCl) seeds are polluted with fungi, mostlyAspergillus flavus, A. niger, Penicillium chrysogenum, P. corylophilum andRhizopus stolonifer. Contamination of popcorn with the fungi is about 10 times higher than in the other foods. These fungi, common also on unsalted seeds, are significantly inhibited in seeds (30% moisture content) treated with 9–21% NaCl. The halotolerantA. wentii represents the main fungus recovered from seeds treated by 15–21% NaCl. 9% NaCl stimulated emodin production byA. wentii on peanut and citrinin production byP. chrysogenum on popcorn and sunflower. Aflatoxin, citrinin and emodin production on popcorn persisted up to 15% NaCl. Popcorn is thus strongly susceptible to fungal invasion and toxin pollution. The halotolerance ofA. wentii was confirmed by its strong permanent growth in liquid medium at up to 15% NaCl. At 3% NaCl the mycelial growth and nitrogen content increased while the level of emodin and lipid production decreased. CO₂ evolution strongly increased at 9–15% NaCl as a characteristic ofA. wentii salt tolerance. Emodin inhibited seed viability and the inhibition dose for 50% reduction (LD₅₀) was 65 mg/L for popcorn and 45 mg/L for sunflower.     

FOURIER TRANSFORM INFRARED SPECTROSCOPY OF MICROALGAE AS A NOVEL TOOL FOR BIODIVERSITY STUDIES,SPECIES IDENTIFICATION,AND THE ASSESSMENT OF WATER QUALITY1

Fourier transform infrared (FTIR) spectrometry was used to study the spectral features of 12 eukaryotic and two prokaryotic species of microalgae. The algae were cultured in liquid media containing either NO₃^? or NH₄⁺ as the sole N‐source; for the NH₄⁺ treatment, the algae were subjected to short‐term (24 h) or long‐term (1 month) incubations; for the hypersaline species, cells were also grown in the presence of 2 M NaCl. Over 500 spectra, acquired from at least three distinct cultures for each species, in each growth regime, were subjected to hierarchical cluster analysis (HCA) and were successfully separated according to their taxonomy, showing that the overall spectra were characteristic of each species and that this technique could be fruitfully employed to separate microalgal species living in a similar condition (as would be the case for a natural assemblage). In addition, in most cases, it was possible to differentiate between algae subjected to different growth treatments although belonging to the same species. We also demonstrated that it is possible to accurately identify species and determine the nutritional status of their environment of origin (e.g., N‐source), provided that suitable FTIR spectral libraries are available. This study aims to provide the basis for the development of rapid, easy, and inexpensive methods for the evaluation of biodiversity in natural phytoplankton samples and to monitor the water quality of natural environments.     

GROWTH OF PHELLINUS (PORIA) WEIRII ON DIFFERENT VITAMINS AND CARBON AND NITROGEN SOURCES

Mycelial growth of Phellinus weirii (Murr.) Gilb., a root pathogen of conifers in the Pacific Northwest, was studied in defined liquid media with different carbon and nitrogen sources and vitamins. The fungus grew significantly more on glucose, xylose, maltose, or fructose than on other carbon sources. Starch did not support growth. Maximum growth occurred in 4 weeks on all carbon sources except dextrin and sucrose, with which maximum growth occurred in 3 weeks. Of the nitrogen sources, peptone supported the best growth of the fungus; glutamic acid, serine, aspartic acid, alanine, leucine, ammonium sulphate, and urea supported significantly better growth than tyrosine, arginine, methionine, threonine, and glycine. Potassium nitrate, phenylalanine, sodium nitrate, lysine, proline, and cysteine inhibited growth. Thiamine hydrochloride was not absolutely required for growth of P. weirii, but better growth was obtained with its addition. The fungus showed no significant responses to a range of other vitamins.     

Wallemia peruviensis sp. nov., a new xerophilic fungus from an agricultural setting in South America

We obtained four isolates of the xerophilic genus Wallemia from the rooftop of a house made of red brick and cement in an agronomic field planted with common beans and maize in Pachacamac, Lima, Peru. Bayesian phylogenetic analysis with rDNA gene sequences showed these Wallemia isolates form a distinct and strongly supported clade closely related to W. hederae. We examined the macro and micromorphology, growth rate and production of exudates of isolates on media containing different amounts of glucose and NaCl (water activity from 0.9993 to 0.8480). Their chaotropic and kosmotropic tolerance were tested on media with multiple molar concentrations of MgCl₂ and MgSO₄ (water activity from 0.9880 to 0.7877). Isolates are xerophilic and halotolerant, growing on 17% NaCl-supplemented media (water activity = 0.8480). Maximum concentrations of MgCl₂ and MgSO₄ at which growth was observed were 1.7 and 3.5 M, respectively. Isolates were shown to represent a novel species, described as Wallemia peruviensis sp. nov. In contrast to W. hederae, W. peruviensis does not produce exudates on malt extract agar + 17% NaCl media. An updated dichotomous key to Wallemia species is provided. This is the first new species of Wallemia described from South America and the first association of a Wallemia species with an agricultural environment on this continent.

     

Effect of water activity adjusted with different solutes on growth and lactic acid production byLactobacillus helveticus

Effect of water activity (a _w) adjusted with NaCl or glycerol on the growth and metabolism ofLactobacillus helveticus var.pragensis at 40°C was demonstrated by growth curves (generation time) and changes in the degree of acidity of a milk medium expressed as lactic acid content. NaCl-regulateda _w of 0.970 inhibited growth and acid production completely. The same, glycerol-regulateda _w of 0.970 increased the generation time only from 33 min (a _w=0.994) to 67 min which was less than the generation time at the highera _w of 0.982 adjusted with NaCl (103 min). Glycerol-regulateda _w of 0.970 did not decrease the acid production (2.6% lactic acid). Ata _w of 0.951, the acid production was decreased by 39% compared with the values found in milk media with the original, unadjusteda _w of 0.994, after the same time of incubation. Media witha _w adjusted to the same values with NaCl or glycerol do not influence the growth and acid production ofL. helveticus in the same way. In contrast to glycerol, NaCl has a strong effect.     

A NUTRITIONAL ANALYSIS OF A SUBLITTORAL DIATOM ASSEMBLAGE EPIPHYTIC ON ENTEROMORPHA FROM A LONG ISLAND SALT MARSH1,2

The diatom population structure of a salt marsh epiphytic community growing on Enteromorpha intestinalis was studied at one station throughout the summer. A total of 218 species or varieties were recognized. Six species— Fragilaria construens, Cocconeis scutellum, Cocconeis placentula, Achnanthes hauckiana varieties, Achnanthes pinnata, and Amphora coffeaeformis (var. acutiuscula)–dominated the Enteromorpha epiphytic community during the summer months and comprised ∼ 40% of the total populations. Melosira nummuloides, Opephora martyi, Synedra fasiculata var. tabulata, S. affinis, Navicula platyventris, and N. pavillardi were also very common species (13% of the total population). The distribution of many species in the community was seasonal. A series of differential media have been developed which are effective as tools for the isolation and nutritional characterization of the algae and bacteria from the community. Many diatom species can be recognized by their colony type or growth pattern on solidified media. A key and illustrated plates aid in identification. Changes in population structure of the community were reflected by changes in the nutritional patterns as judged by differences in the growth of diatoms and bacteria on the differential media tested. The nutritional requirements, selectivity, and rank order of media for individual species is given. Almost half of the colony types (32–33) grew on either unenriched seawater from the collection site or basal synthetic seawater. Erdschreiber was a poorer medium for the isolation of algae than seawater alone. Few algal colonies grew on either marine nutrient agar or trypticase soy agar. Nitrite inhibited algal growth. Media enriched with thiamine, biotin, or nitrate, phosphate, and B₁₂ were stimulatory. Soil extract or an acetone extract of Enteromorpha tripled growth; a complex vitamin mixture, or glycerol, or mannitol, or an autoclaved extract of Enteromorpha doubled colony numbers. The greatest numbers of diatom types (43) and total colonies (655) were recovered on media enriched with acetone extract of Enteromorpha, suggesting a possible nutritional relationship between Enteromorpha and its epiphytes. The representativeness of the isolations from the natural community as a function of time was also considered. Concentrations of selected dissolved potential growth-stimulating nutrients were measured within and external to Enteromorpha communities. We conclude that the algal assemblage growing epiphytically on Enteromorpha intestinalis has a diverse auxotrophic profile which contributes to the productivity and stability within this important component of the epiphytic community and that much of the organic substrates used by individual species originate within and are recycled among the community members.     

Biology of a problematic marine fungus,dermocystidium sp.

Summary The life cycle of a nonfilamentous marine phycomycete, Dermocystidium sp., is described from its growth in axenic culture under controlled conditions. Reproduction occurs by vegetative means involving a process of cell enlargement and ecdysis modifiable by the methods of cultivation. Zoospores and sexuality were not observed. The results of inoculation studies with algae, phanerogam debris, and molluscs suggest that the fungus may be a free-living saprophyte in coastal waters.     

Characterization of aMethanosarcina strain isolated from goat feces,and that grows on H2-CO2 only after adaptation

AMethanosarcina species, designated strain ChGul, was isolated from goat feces; this is the first fully described pure culture ofMethanosarcina obtained from feces. Antigenic fingerprinting suggests that isolate ChGul is a new immunotype. The mol% G + C content of DNA was 42.2%. Strain ChGul grew on methanol, methylamines, and acetate in a minimal salts medium. It grew on H₂-CO₂ only after adaptation. Growth occurred as a milky-white suspension and contained cells mostly in doublets and quadruplets of irregular cocci; many cells contained phase bright spots typical of gas vacuoles. The isolate did not grow on formate, or CO₂ plus isopropanol, ethanol, or acetone as substrates and did not produce methane from formate. The optimum growth temperature was 35–37°C, and optimum pH was 6.2–6.8. ChGul is unusually sensitive to sulfide and has low tolerance for NaCl. Optimal levels of total sulfide and NaCl for growth were 0.5 mM and 20–40 mM, respectively. Since ChGul requires adaptation for growth on H₂-CO₂ and cannot use formate, it may be restricted to methylotropic or acetoclastic methanogenesis in the rumen, a function not observed in previously isolated rumen methanogens that use H₂-CO₂ and formate. Our work suggests that improper NaCl and sulfide concentrations, and cell lysis, may have made isolation of rumenMethanosarcina difficult in the past. It also underscores the need to evaluate feed compositions and media components for most probable number studies, with respect to NaCl and sulfide levels, to understand the role ofMethanosarcina in the rumen.